Jiro Takeo

Two Distinct Isoforms of cDNA Encoding Rainbow Trout Androgen Receptors

Androgens play an important role in male sexual differentiation and development. The activity of androgens is mediated by an androgen receptor (AR), which binds to specific DNA recognition sites and regulates transcription. We describe here the isolation of two distinct rainbow trout cDNA clones, designated rtAR-α and rtAR-β, which contain the entire androgen receptor coding region. Comparison of the predicted amino acid sequence of rtAR-α to that of rtAR-β revealed 85% identity. Interestingly, despite this high homology, rtAR-α activated transcription of an androgen-responsive reporter gene in co-transfection assays, but rtAR-β did not. These results suggest that rainbow trout contains two distinct isoforms of androgen receptors whose functions differ. The region of rtAR-β responsible for its inactivity was mapped to its ligand binding domain by analyzing chimeras of the rtAR-α, rtAR-β, and rtGR-I (glucocorticoid) receptors. Alteration of any one of three out of four segments within this domain restored activity. Extracts made from COS-1 cells transfected with an rtAR-α expression plasmid produced a high level of [3H]mibolerone binding, whereas no binding was observed by extracts of cells transfected with an rtAR-β expression plasmid. These data demonstrate that the lack of transactivation activity of rtAR-β is due to its inability to bind hormone.

Fish glucocorticoid receptor with splicing variants in the DNA binding domain

Here we describe the isolation of a rainbow trout cDNA containing an entire GR coding region. Although the encoded protein is highly homologous to other GRs, especially in its DNA binding domain, it contains a nine amino acid insertion between the two zinc fingers. This novel form is found in all rainbow trout tissues examined; however, the testis also contains a splice variant lacking this insert, making it completely continuous to other GRs. In transient transfection assays of cultured cells, the two rainbow trout GR variants activated transcription from the glucocorticoid‐responsive mouse mammary tumor virus promoter to comparable levels.

Pharmacological detection of AMPA receptor heterogeneity by use of two allosteric potentiators in rat hippocampal cultures

In order to examine whether a recently developed allosteric potentiator for AMPA receptors, 4‐[2‐(phenylsulphonylamino)ethylthio]‐2,6‐difluoro‐phenoxyacetamide (PEPA), can be utilized as an indicator of AMPA receptor heterogeneity, the action of PEPA upon the increase of intracellular free calcium ion concentration ([Ca2+]i) elicited by AMPA was investigated in rat hippocampal cultures, and the action was compared with that of cyclothiazide, a well characterized allosteric modulator of AMPA receptors. PEPA dose‐dependently potentiated AMPA‐induced increase of [Ca2+]i. In 90% (72 out of 80) of the cells in which cyclothiazide acts, PEPA potentiated the increased [Ca2+]i induced by AMPA with pronounced cell‐to‐cell variation in rat hippocampal cultures. The ratio of the potentiation by PEPA to the potentiation by cyclothiazide (P/C ratio) also varied with cells between 0 and 2.15. It was found that the cultured hippocampal cells consisted of multiple populations with different P/C ratios. Among them two populations exhibited characteristic P/C ratios; low (0 to 0.15; 27 out of 80 cells, 34%) and high (2.00; 1 out of 80 cells, 1%) P/C ratios. The P/C ratios of the other populations were between 0.25 and 1.20, and these cells constituted 65% (52 out of 80 cells) of the cells tested. Reverse transcriptase‐polymerase chain reaction analysis suggested that GluR2‐flip, GluR1‐flip, GluR2‐flop, and GluR1‐flop were abundantly expressed (in this rank order) in the cultures used. In Xenopus oocytes expressing GluR1, GluR3, or these subunits plus GluR2, the potentiation of AMPA response by PEPA and by cyclothiazide varied with subunit and splice‐variant combinations, and the P/C ratio was between 0.19 and 2.20. Oocytes with low P/C ratios (0.19 to 0.50) and low sensitivity to PEPA potentiation (1.9 fold to 6.41 fold) were those expressing flip variants predominantly, and oocytes with high P/C ratios (1.8 to 2.2) were those expressing flop variants predominantly. Oocytes with intermediate P/C ratios (0.51 to 1.20) were those expressing various combinations of flip and flop variants, and it was impossible to specify the relative abundance of flip and flop variants in these cells. Therefore, the P/C ratio can be used to infer subunit/splice variant expression only when the ratio is low or high. These results suggest that the potentiation by PEPA alone reveals cell‐to‐cell heterogeneity of AMPA receptors, but a comparison of the actions of PEPA and cyclothiazide further facilitates the detection of the heterogeneity.

Alstiphyllanines E–H, picraline and ajmaline-type alkaloids from Alstonia macrophylla inhibiting sodium glucose cotransporter

Three new picraline-type alkaloids, alstiphyllanines E-G (1-3) and a new ajmaline-type alkaloid, alstiphyllanine H (4) were isolated from the leaves of Alstonia macrophylla together with 16 related alkaloids (5-20). Structures and stereochemistry of 1-4 were fully elucidated and characterized by 2D NMR analysis. Alstiphyllanines E and F (1 and 2) showed moderate Na(+)-glucose cotransporter (SGLT1 and SGLT2) inhibitory activity. A series of a hydroxy substituted derivatives 21-28 at C-17 of the picraline-type alkaloids have been derived as having potent SGLT inhibitory activity. 10-Methoxy-N(1)-methylburnamine-17-O-veratrate (6) exhibited potent inhibitory activity, suggesting that the presence of an ester side chain at C-17 may be important to show SGLT inhibitory activity. Structure activity relationship of alstiphyllanines on inhibitory activity of SGLT was discussed.

Cyclic diarylheptanoids as Na+-glucose cotransporter (SGLT) inhibitors from Acer nikoense.

Two cyclic diarylheptanoids, acerogenins A (1) and B (2) have been isolated from the bark of Acer nikoense as inhibitors of Na(+)-glucose cotransporter (SGLT). Acerogenins A (1) and B (2) inhibited both isoforms, SGLT1 and SGLT2. Structure-activity relationship of acerogenin derivatives on inhibitory activity of SGLT as well as conformational analysis of 1 and 2 on the basis of J-resolved HMBC spectra and X-ray analysis were discussed.

A cDNA Encoding Fish Fibroblast Growth Factor-2, Which Lacks Alternative Translation Initiation

Here, we describe the isolation of a rainbow trout cDNA clone that contains the entire fibroblast growth factor-2 (FGF-2; basic FGF) coding region. Interestingly, the rainbow trout cDNA contains a translation stop codon just upstream of the primary initiating methionine codon and so cannot give rise to the longer forms of FGF-2 that are produced in mammals by alternative translation initiation at leucines farther upstream. Transfection of human FGF-2 cDNA into fish cells shows that fish cells can initiate protein synthesis at an upstream leucine CUG codon; surprisingly, however, synthesis is initiated only at the most 5′ CUG and not at the two subsequent CUG codons or the methionine AUG codon also used in mammalian cells. Like other FGF-2 proteins, bacterially produced rainbow trout FGF-2 protein binds tightly to heparin-Sepharose and also promotes the proliferation of fibroblast cells. However, the protein differs from all others previously identified at amino acids 121-123, which are part of the proposed highly conserved receptor-binding domain. Comparisons of the efficacies of recombinant wild-type and mutant rainbow trout FGF-2 proteins demonstrate that these three amino acids are critical to the activity of FGF-2.

Modulation of Fear Memory by Dietary Polyunsaturated Fatty Acids via Cannabinoid Receptors

Although the underlying mechanism remains unknown, several studies have suggested benefits of n-3 long-chain polyunsaturated fatty acid (PUFA) for patients with anxiety disorders. Elevated fear is thought to contribute to the pathogenesis of particular anxiety disorders. The aim of the present study was to evaluate whether the dietary n-3 to n-6 PUFA (3:6) ratio influences fear memory. For this purpose, the effects of various dietary 3:6 ratios on fear memory were examined in mice using contextual fear conditioning, and the effects of these diets on central synaptic transmission were examined to elucidate the mechanism of action of PUFA. We found that fear memory correlated negatively with dietary, serum, and brain 3:6 ratios in mice. The low fear memory in mice fed a high 3:6 ratio diet was increased by the cannabinoid CB1 receptor antagonist rimonabant, reaching a level seen in mice fed a low 3:6 ratio diet. The agonist sensitivity of CB1 receptor was enhanced in the basolateral nucleus of the amygdala (BLA) of mice fed a high 3:6 ratio diet, compared with that of mice fed a low 3:6 ratio diet. Similar enhancement was induced by pharmacological expulsion of cholesterol in the neuronal membrane of brain slices from mice fed a low 3:6 ratio diet. CB1 receptor-mediated short-term synaptic plasticity was facilitated in pyramidal neurons of the BLA in mice fed a high 3:6 ratio diet. These results suggest that the ratio of n-3 to n-6 PUFA is a factor regulating fear memory via cannabinoid CB1 receptors.

Lorneic Acids, Trialkyl-Substituted Aromatic Acids from a Marine-Derived Actinomycete

A marine-derived actinomyces strain (NPS554) isolated from a marine sediment sample collected from Miyazaki Harbor, Japan, at a depth of 38 m yielded two trialkyl-substituted aromatic acids, lorneic acid A (1) and lorneic acid B (2). The structures of the lorneic acids, which were elucidated by spectroscopic analysis, differed only in the side-chain, which contained either a conjugated double bond or a benzylic alcohol. Their structural differences affected inhibition activities against phosphodiesterase 5.

Alleviation of conditioned fear in mice fed with lipid extracts of Euphausia superba

outgrowth by As. In contrast to the striking effect on neurite outgrowth, overexpression of GluR2 could not rescue the mortality of neurons in the culture at concentration above 2 M As. These results suggest the possibility that suppression of neurite outgrowth by As in primary cortical neurons occurs through impact on glutamate transmission, which might be independent of the effect on cell death. Research fund: Supported by National Institute for environmental studies (0406AG337).